Enhanced oxidative activities of cytochrome P450Rhf from Rhodococcus sp. expressed using the hyper-inducible expression system.

Bacterial cytochrome P450 enzymes catalyze the oxidative biotransformation of various types of compounds. Although the functional expression of Actinomycetes P450 in a closely related heterologous host can serve as a useful biocatalyst in whole-cell biotransformation assays, the co-expression of an electron transfer partner is required. To overcome this limitation, P450Rhf from Rhodococcus sp. NCIMB 9784 is an ideal candidate, because it is fused to a reductase domain at the C terminus and does not require an electron transfer partner. Here, we cloned P450Rhf into the hyper-inducible expression vector pSH19 in Streptomyces lividans TK24 for developing an efficient whole-cell biotransformation system with bacterial P450. The recombinant strain displayed high conversion activity (79.1%) of 7-ethoxycoumarin to 7-hydroxycoumarin after 48 h, and the observed activity was markedly higher than those for 7-methoxycoumarin and 7-propoxycoumarin used as substrates. We next screened several commercially available substrates possessing an ethyl phenyl ether moiety, which is also present in 7-ethoxycoumarin, and found that 4'-ethoxy-2'-hydroxyacetphenone was almost completely dealkylated (95.0%), and that 7-ethoxy-4-methylcoumarin was converted to two products, 7-hydroxy-4-methylcoumarin and 7-ethoxy-4-hydroxymethyl-coumarin. Our research suggests that enhancement of heterologous P450 expression using the pSH19 system in whole-cell biotransformation assays is valuable for identifying novel substrates of P450, as well as for obtaining high yields of conversion products.

Strain selection and scale-up fermentation for FR901379 acylase production by Streptomyces sp. no. 6907.

Micafungin (FK463) is a widely used treatment for life-threatening, deep-seated fungal infections. It is an echinocandin-like lipopeptide derived from the chemical modification of deacylated FR901379, a type of lipopeptide antibiotic produced by Coleophoma empetri F-11899. The palmitoyl moiety of FR901379 is deacylated by FR901379 acylase produced by Streptomyces sp. no. 6907. In this study, our goal was to generate an improved strain of Streptomyces sp. no. 6907 capable of hyperproducing the FR901379-acylase enzyme. To accomplish this goal, modified strains of Streptomyces sp. no. 6907 were generated using UV-irradiation mutagenesis, and strain selection was performed using an agar-plate screening method to efficiently select an acylase-hyperproducing strain. Three marker indices were shown to correlate with elevated acylase production: decreased candidacidal activity of FR901379, decreased proteolytic activity on skim milk, and phenotypic characteristics. Cloning and subsequent sequencing of the acylase gene from the hyperproducing mutant revealed no mutations in either the acylase structural gene or the 5'-flanking region required for gene expression. The growth medium was also modified to maximize acylase production. We successfully increased acylase activity approximately 65-fold, compared with the original growth conditions (wild strain cultured in the original unmodified medium). To minimize formation of excess foam during the fermentation process, we optimized the parameters of agitation speed, as calculated from the discharge flow rate. Using our improved strain and the optimized medium and growth conditions, we have developed an improved and highly reproducible method for stable large-scale production of FR901379-acylase.

Cloning and expression of the FR901379 acylase gene from Streptomyces sp. no. 6907.

FR901379 acylase, an enzyme that catalyzes the hydrolysis of the palmitoyl moiety of the antifungal lipopeptide FR901379, was purified from the culture broth of Streptomyces sp. no. 6907 (FERM BP-5809), revealing the 80 kDa, two-subunit heterodimeric protein characteristic of the ß-lactam acylase family. Using oligodeoxyribonucleotide primers constructed on the basis of the N-terminal amino acid sequence of each purified subunit, the gene was identified from a cosmid library of Streptomyces sp. no. 6907 DNA. The deduced 775 amino acid sequence corresponded to a single polypeptide chain containing two subunits, and it shared 41.7% identity with aculeacin A acylase from Actinoplanes utahensis NRRL12052. FR901379 acylase activity was found to be 250-fold higher in the recombinant Streptomyces lividans 1326 carrying the cloned gene than in the original Streptomyces sp. no. 6907 strain.

[Drug development from natural fermentation products: establishing a manufacturing process which maximizes the potential of microorganisms].

Natural fermentation products have long been studied as attractive targets for drug discovery due to their amazing diverse, complex chemical structures and biological activities. As such, a number of revolutionary drugs developed from natural fermentation products have contributed to global human health. To commercialize a drug derived from natural fermentation products, an effective chemical entity must be identified and thoroughly researched, and an effective manufacturing process to prepare a commercial supply must be developed. To construct such a manufacturing process for tacrolimus and micafungin, the following studies were conducted: first, we focused on controlling the production of the tacrolimus-related compound FR900525, a fermentation by-product of tacrolimus which was critical for quality assurance of the drug substance. FR900525 production was reduced by using a mutant strain which produced more pipecolic acid, the biosynthesis material of tacrolimus, than the original strain. Then, to optimize the fermentation process of FR901379, an intermediate of micafungin, a fed-batch culture was adopted to increase FR901379 productivity. Additionally, FULLZONE(TM) impeller was installed into the scaled-up fermenter, reducing the agitation-induced damage to the mycelium. As a result, the mycelial form changed from filamentous to pellet-shaped, and the air uptake rate during fermentation was drastically improved. Finally, we conducted screening for FR901379 acylase-producing microorganisms, as FR901379 acylase is necessary to manufacture micafungin. We were able to easily discover FR901379 acylase-producing microorganisms in soil samples using our novel, convenient screening method, which involves comparing the difference in antibiotic activity between FR901379 and its deacylated product.

Bioconversion of AS1387392: bioconversion studies involving Amycolatopsis azurea JCM 3275.

We screened actinomycetes capable of converting AS1387392 to AS1429716 and identified those strains capable of hydroxylation. Amycolatopsis azurea JCM 3275 was found to be a particularly efficient strain, capable of converting AS1387392 to AS1429716, with a yield of 44% after 9 h. This strain can metabolize not only the hydroxylation of phenylalanine at the meta and para positions but also the reduction of hydroxyketones, as shown by the isolation of bioconversion products. Examination of more suitable conversion conditions showed that pH 7.8 and 25 °C were the optimum pH and temperature for bioconversion, respectively. We also demonstrated the effect of carbon and nitrogen sources in the culture media on hydroxylation. Using this strain, we were able to efficiently produce AS1429716 as a chemical template. Further derivatization studies may provide more effective, safer immunosuppressants than those that are currently on-market.

Cloning and heterologous expression of P450Um-1, a novel bacterial P450 gene, for hydroxylation of immunosuppressive agent AS1387392.

Biotransformation technology involving enzymatic modification of original substrates by organisms such as microbes is a valuable tool in improving pharmacokinetics or physicochemical properties of the base compounds. The fungal metabolite AS1387392 is a histone deacetylase inhibitor with potential as a therapeutic immunosuppressant. However, its paucity of functional groups, essential to synthesizing derivatives, is a drawback. Amycolatopsis azurea JCM-3275 catalyzed hydroxylation of AS1387392 to AS1429716, which may facilitate the synthesis of more derivatives by the additional hydroxyl moiety present in AS1429716. This reaction was inhibited by cytochrome P450 inhibitor metyrapone, indicating that cytochrome P450 may be responsible for the transformation. Degenerate PCR primers were subsequently constructed and used to clone genes encoding cytochrome P450 from the genomic DNA of A. azurea JCM-3275. We cloned an entire novel P450 gene (1209 bp) and named it P450Um-1. Its deduced amino acid sequence was homologous with that of the CYP105 subfamily. Further cloning of the upstream region, which may contain the native promoter site, was followed by insertion of the open reading frame with the upstream area into Streptomycetes high copy vector pIJ702, giving the expression plasmid pNUm-1. P450Um-1 was specifically expressed in Streptomyces lividans TK24, and this recombinant strain converted AS1387392 to AS1429716 without any redox partners. These results show that P450Um-1, a novel bacterial P450, catalyzed hydroxylation of AS1387392 to AS1429716. This resultant recombinant strain is expected to be an efficient biocatalyst with application to more suitable redox systems than those tested here.

Bioconversion of AS1387392: screening and characterization of actinomycetes that convert AS1387392 to AS1429716.

AS1387392 was a novel and powerful histone deacetylase inhibitor with an excellent oral absorption profile, but this compound was lacking in active moieties, which are essential to synthesize more derivatives. In our screening program to identify actinomycetes capable of converting AS1387392 to AS1429716, which has an active moiety to synthesize more derivatives, we identified 12 strains capable of efficient hydroxylation. Results of phylogenetic analysis of 16S rDNA sequences suggested that these strains belonged to the genera Lentzea, Saccharopolyspora, Sphaerisporangium and Amycolatopsis. Morphological and chemical characteristics as well as results of phylogenetic analysis suggested that strain No. 7980 was a new species belonging to the genus Amycolatopsis, according to the FASTA search result of 16S rDNA gene sequence. Using these strains, we can easily produce AS1429716 as a chemical template for further chemical modifications, which may provide more effective and safer immunosuppressant.

AS1387392, a novel immunosuppressive cyclic tetrapeptide compound with inhibitory activity against mammalian histone deacetylase.

The novel immunosuppressant AS1387392 has been isolated from Acremonium sp. No. 27082. This compound showed a strong inhibitory effect against mammalian histone deacetylase and T-cell proliferation. Further, AS1387392 showed a good oral absorption, and its plasma concentration was higher than that of FR235222, an analog of AS1387392 that inhibited histone deacetylase previously reported. Given these findings, AS1387392 may represent an important new lead in developing immunosuppressant.

Screening and characterization of microorganisms with FR901379 acylase activity.

A simple and rapid screen was performed for microorganisms producing cyclic lipopeptide acylase, an enzyme that deacylates the acyl side chain of FR901379 to yield a cyclic peptide, FR179642, which is an important intermediate in producing micafungin. Among approximately 3800 newly isolated strains from soil samples, 5 microorganisms expressing high acylase activity were selected and classified, 3 as Streptomyces spp. and 2 as filamentous fungi. This is the first time that these strains have been identified as cyclic lipopeptide acylase producers. Both fungi and streptomycetes showed differing optimum pH and temperature profiles for acylase activity.

Discovery of anti-varicella zoster virus activity of polyether antibiotic CP-44161.

In search for new anti-varicella zoster virus (VZV) compounds with new mechanism of action, we applied a DNA hybridization assay (dot blot method) for screening. Using this method, we screened microbial products and found the polyether compound CP-44161 from the culture broth of an actinomycete strain. CP-44161 was previously reported as an anticoccidal agent, but there has been no claim of its antiviral activities. CP-44161 showed strong anti-VZV activity against pOka strain by plaque reduction assay. Moreover, CP-44161 showed lower cytotoxicity than other antiviral polyethers, such as monensin and nigericin. Its better safety margin and strong anti-VZV properties make it a good candidate for a new anti-VZV agent.

Anti-herpes virus activity of polyether antibiotic CP-44161 in vivo.

In the previous study, we discovered a polyether antibiotic CP-44161, which was reported earlier as an anticoccidal agent, as an anti-varicella zoster virus compound. In this study, we demonstrated that CP-44161 had a very strong and broad anti-herpes virus activities against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in vitro. To determine the antiviral activity of CP-44161 in vivo, we examined its effect on the cutaneous HSV-2 infection model in Balb/c mice. CP-44161 showed inhibitory effect on lesion development as well as acyclovir (ACV) when the treatment was started from day 3. Meanwhile, in case the start of treatment was delayed until day 4, when ACV was no longer effective, the effectiveness of CP-44161 still remained. In this model, CP-44161 also showed inhibitory effect on the proliferation of HSV-2 DNA in dorsal root ganglia. This is the first article to report that polyether antibiotics can be effective on viral infection in vivo.

Osteopontin is dispensable for protection against high load systemic fungal infection.

Osteopontin (OPN) is a multi-functional cytokine which is involved in the pathogenesis of autoimmune disease. We previously reported that thrombin-cleaved form of OPN plays a pathogenic role in murine model of rheumatoid arthritis (RA) by using neutralizing antibody (M5) reacting against the cryptic epitope within OPN, exposed by thrombin cleavage of OPN. It has been shown that OPN-deficient mice are susceptible to various infections, demonstrating the protective role of OPN against various infectious diseases. However, it remains to be clarified whether and how OPN is involved in protection against systemic fungal infection. In a murine model of systemic fungal infection, OPN-deficient mice showed the increase in the susceptibility to low load, but not to high load fungal infection, indicating the protective of OPN against mild or severe forms of infections. However, mice treatment with M5 antibody did not alter the susceptibility to both high and low load fungal infection. These experiments suggest that in sharp contrast to the complete abrogation of OPN expression in OPN-deficient mice, the neutralization of OPN by antibody against thrombin-cleaved form of OPN does not interfere with the host defense against high and low load fungal infection. These findings suggest that the neutralizing antibody which is specific for the epitope of thrombin-cleaved OPN may become an attractive therapeutic means for the treatment of RA without interfering host defense system.

Neuroprotective efficacy of FR901459, a novel derivative of cyclosporin A, in in vitro mitochondrial damage and in vivo transient cerebral ischemia models.

The immunosuppressant cyclosporin A (CsA) has been shown to exert potent neuroprotective effects, possibly via the inhibition of calcineurin and mitochondrial permeability transition pore formation. Here, we investigated the neuroprotective profile of a novel derivative of CsA, FR901459, by evaluating its effects against in vitro mitochondrial damage and in vivo brain damage in transient global or focal cerebral ischemia models, in comparison with those of CsA. Efficacy of calcineurin inhibition was estimated from its immunosuppressive effect on the mixed lymphocyte reaction. Results showed that the immunosuppressive effect of FR901459 was approximately 7-fold less potent than that of CsA. In contrast, FR901459 suppressed Ca(2+)-induced mitochondrial swelling measured in isolated liver mitochondria with greater potency than CsA. Further, FR901459 showed approximately 30-fold greater neuroprotective potency than CsA against neuronal cell damage induced by thapsigargin in SH-SY5Y cells. In a transient global cerebral ischemia model in gerbils, FR901459 showed the dose-dependent suppression of neuronal cell death, while FR901459 was less efficacious than CsA. In a rat transient focal ischemia model, FR901459 tended to reduce brain damage on both intravenous injection as well as intracerebroventricular infusion, but with less efficacy than CsA which significantly reduced the damage. These findings suggest that FR901459 exerts a potent neuroprotective effect by inhibiting mitochondrial damage in vitro, but that in in vivo transient cerebral ischemia, its immunosuppressive component which possibly acts via the inhibition of calcineurin may play a more important role in attenuating brain damage than its inhibitory effect against mitochondrial damage.

FR209602 and related compounds, novel antifungal lipopeptides from Coleophoma crateriformis no.738. I. Taxonomy, fermentation, isolation and physico-chemical properties.

Novel antifungal lipopeptides, FR209602, FR209603 and FR209604, were isolated from the fermentation broth of a fungal strain No. 738 which was identified as Coleophoma crateriformis from morphological and physiological characteristics. The antibiotics were purified by solvent extraction, HP-20, YMC-ODS and silica gel column chromatography and lyophilization. These compounds were structurally similar to FR901379 previously reported by ourselves which had a sulfate residue in the cyclic peptide portion.

FR209602 and related compounds, novel antifungal lipopeptides from coleophoma crateriformis no. 738. II. In vitro and in vivo antifungal activity.

The biological activities of the novel echinocandin-like lipopeptides, FR209602, FR209603 and FR209604, were evaluated. These compounds showed antifungal activity against Candida albicans and Aspergillus fumigatus attributed to inhibition of 1,3-beta-glucan synthesis. The minimum effective concentrations of these compounds against C. albicans and A1. fumigatus ranged from 0.02 to 0.04 microg/ml by microbroth dilution assay, and the IC50 values on C. albicans 1,3-beta-glucan synthase were 0.49, 0.64 and 0.72 microg/ml, respectively. FR209602 and FR209603 showed good efficacy by subcutaneous injection against C. albicans in a murine systemic infection model, with ED50 values of 2.0 and 1.9 mg/kg, respectively.

FR227673 and FR190293, novel antifungal lipopeptides from Chalara sp. No. 22210 and tolypocladium parasiticum No. 16616.

Novel antifungal lipopeptides, FR227673 and FR190293, were isolated from the fermentation broths of fungal strains Chalara sp. No. 22210 and Tolypocladium parasiticum No. 16616, respectively. These compounds have the same cyclic peptide nuclear structure as FR901379, with different side chains, and showed antifungal activity against Aspergillus fumigatus and Candida albicans attributed to inhibition of 1,3-beta-glucan synthesis.

FR220897 and FR220899, novel antifungal lipopeptides from Coleophoma empetri no. 14573.

Novel antifungal lipopeptides, FR220897 and FR220899, were isolated from the fermentation broth of a fungal strain No. 14573. This strain was identified as Coleophoma empetri No. 14573 from morphological and physiological characteristics. FR220897 and FR220899 showed antifungal activities against Aspergillus fumigatus and Candida albicans attributed to inhibition of 1,3-beta-glucan synthesis. Furthermore, FR220897 was effective in a murine model of systemic candidiasis.

Oxidative activities of heterologously expressed CYP107B1 and CYP105D1 in whole-cell biotransformation using Streptomyces lividans TK24.

Cytochrome P450 enzymes are a major class of biocatalysts related to the oxidative metabolism of many drugs, assisted by electron transfer partners. The functional expression of the P450 gene in a heterologous host will lead to efficient biotransformation and biodegradation, which are useful in pharmaceutical improvement or environmental cleanup. The soluble cytochrome P450 monooxygenase systems CYP105D1 and CYP107B1 involved in the biotransformation of some xenobiotics, such as secondary metabolites or environmental pollutants, were expressed in Streptomyces lividans TK24 with the Streptomyces expression vector pIJ6021. In whole-cell biotransformation assay using these recombinant strains, the oxidative dealkylation of 7-ethoxycoumarin was detected without any foreign redox partners in the case of CYP107B1, while the activity of CYP105D1 was not monitored until this gene was coexpressed with the ferredoxin gene located downstream of the CYP105D1 gene, and the ferredoxin reductase gene SCF 15.02 from Streptomyces coelicolor A3(II). This result suggests that CYP107B1 is capable of utilizing an endogenous electron transfer partner from the host but not CYP105D1, and that CYP105D1 is complemented by some redox partner imported from closely related strains.

FR258900, a novel glycogen phosphorylase inhibitor isolated from Fungus No. 138354. II. Anti-hyperglycemic effects in diabetic animal models.

A novel glycogen phosphorylase inhibitor FR258900 was isolated from the cultured broth of a fungal strain No. 138354. We examined the hypoglycemic effects of FR258900 in diabetic animal models. FR258900 treatment significantly reduced the plasma glucose concentrations during oral glucose tolerance tests in diabetic mice models, including db/db mice and STZ-induced diabetic mice. Furthermore, FR258900 treatment resulted in rapid decrease in the plasma glucose levels in db/db mice. These improvements in glucose disposal were accompanied by increased liver glycogen contents, suggesting that the glucose lowering effects of FR258900 were attributed to suppressed hepatic glycogen breakdown and increased hepatic glycogen synthesis. Taken together, our results suggest that glycogen phosphorylase is a potentially useful target in new therapies against diabetes.

FR258900, a novel glycogen phosphorylase inhibitor isolated from Fungus No. 138354. I. Taxonomy, fermentation, isolation and biological activities.

FR258900 is a novel glycogen synthesis activator produced by Fungus No. 138354. This compound was isolated from the culture broth by solvent extraction and reverse-phase column chromatography. FR258900 stimulated glycogen synthesis and glycogen synthase activity in primary rat hepatocytes. FR258900 exhibited a potent inhibitory effect on the activity of liver glycogen phosphorylase, suggesting that this compound may activate hepatic glycogen synthesis via glycogen phosphorylase inhibition. Thus, this glycogen phosphorylase inhibitor may be useful in the treatment of postprandial hyperglycemia in type 2 diabetes.